Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.

Research in the field of metallodrugs is continually increasing. However, it is often limited by the poor solubility in water of the metal complexes. To try to overcome this problem, the two new ligands bis-(sodium 3-methoxy-5-sulfonate-salicylaldehyde)thiocarbohydrazone (bis-TCH, Na₂H₄L¹) and bis-(sodium 3-methoxy-5-sulfonate-salicylaldehyde)carbohydrazone (bis-CH, Na₂H₄L²) were synthesized and characterized, both achieving high solubility in water. The speciation of the ligands and their coordinating behaviour towards the biologically relevant Cu(II) and Zn(II) ions were studied spectroscopically and potentiometrically, determining the pK_as of the ligands and the formation constants of the complex species. The monometallic and bimetallic Cu(II) and Zn(II) complexes were isolated, and the single-crystal X-ray structure of [Cu₂(NaHL¹)(H₂O)₇]^.3.5H₂O was discussed. Finally, preliminary studies of the in vitro cytotoxic properties of the new compounds were started on normal (Hs27) and cancer (U937) cell lines. bis-TCH was able to induce a growth inhibition effect between 40% and 45% in both cell lines; bis-CH did not produce a reduction in cell viability in Hs27 cells but revealed mild antiproliferative activity after 72 h of treatment in U937 cancer cells (GI₅₀ = 46.5 ± 4.94 μg/mL). Coordination of the Cu(II) ions increased the toxicity of the compounds, while, in contrast, Zn(II) complexes were not cytotoxic.

Glioblastoma (GBM), a highly malignant brain tumor, arises within a complex microenvironment that plays a critical role in facilitating tumor progression, ensuring survival, and enabling immune evasion, ultimately contributing to therapeutic resistance. Cancer-associated fibrosis is increasingly recognized as a key factor in the tumor pathophysiology, particularly in extracranial cancers, and reported therapeutic strategies in several cancers consist of the current use of the standard-of-care treatment combined with anti-fibrotic drugs. However, it remains unclear how the fibrotic changes associated with the GBM microenvironment contribute to the transformation of GBM from a chemosensitive state to a chemoresistant one. Here, we developed an in vitro model that mimics a fibrosis-like mechanism using the U-87MG GBM cell line. To achieve this, we identified the optimal experimental conditions (i.e., U-87MG cultured in serum-deprivation medium in the presence of recombinant TGF-B1 at 5 ng/mL for 72 h) that effectively induced fibrosis, as suggested by the counter-regulated expression of E- and N-cadherin and sustained levels of α-SMA and collagen I. As expected, U-87MG fibrotic cells were demonstrated to be more resistant to TMZ (predicted EC₅₀ = 35 µM) as compared to the non-fibrotic counterpart (EC₅₀ not achieved here; predicted EC₅₀ = 351 µM). Accordingly, the anti-fibrotic uPAcyclin—a new derivative cyclic compound inspired as a A6 decapeptide drug—showed a significant cytotoxic effect, sensitizing resistant U-87MG fibrotic cells to TMZ. This highlights that targeting fibrosis may help to overcome TMZ resistance in GBM.

Despite extensive efforts to develop new treatments, the prognosis for glioblastoma multiforme (GBM) is extremely unfavorable, urging the identification of new chemotherapeutics. A previous work identified the cyclic decapeptide uPAcyclin as a potent inhibitor of GBM cell migration, matrix invasion and vascular-like structures’ formation, acting through binding to αV integrins and not interfering with cell proliferation or survival. These clearcut activities prompted us to design and test novel derivatives on cultured U87-MG and U251 GBM-MG human cells. With the exception of the residues involved in peptide cyclization, residues were Ala-substituted one by one and the single peptides tested for binding affinity for the αV target integrin, the inhibition of migration, invasion and vasculogenic mimicry. The first screening highlighted peptides with a low binding affinity and low inhibitory ability (Ala4,7,9 derivatives) and peptides with affinity and inhibitory capacity higher than uPAcyclin (Ala2,5,6,8 derivatives). The integration of these results with conformational studies led to the design of the di-substituted variant uPAcyclin. Intriguingly, at least ten-fold greater anti-migratory and anti-invasive effects of the [Ala²,Ala⁵]uPAcyclin variant compared to uPAcyclin were found. The latter variant also exhibited a greater inhibitory potential for vascular-like structures’ formation by matrix-seeded GBM cells. These studies shed light on the functional relevance of single amino acid residues in uPAcyclin and lead to the identification of therapeutically interesting new variants as promising candidates for anti-GBM therapies.